Invaded Freshwater Habitats Research Articles

Environmental conditions influencing the early colonization stage of Ludwigia hexapetala , an aquatic plant recently invasive in Italy

Freshwater ecosystems are among the most susceptible to biological invasions. The South American Ludwigia hexapetala is an aquatic plant that is becoming an increasing threat in many European waterbodies, recently including Italy. This study aimed to define the main parameters influencing the early colonization stage of L. hexapetala by overlapping the percentage cover of this species with environmental parameter data collected at 24 aquatic sites from six waterbodies in north-central Italy. At each site, chemical and physical characteristics of the water (temperature, pH, dissolved oxygen, conductivity, nitrates, phosphates, ammonia, depth, transparency), grain size of the substrate and level of anthropogenic disturbance were evaluated. The results showed that although L. hexapetala prefers shallow, warm, alkaline, moderately rich in ions and nutrients (especially phosphates) and oxygen-poor waters, it can grow in a wide range of environmental conditions. Moreover, as a typical invasive alien species, it spreads opportunistically in disturbed, unstable sites. Thus, L. hexapetala can invade freshwater habitats with different environmental conditions and subjected to anthropogenic disturbance. However, the results suggest that water depth may be a limiting factor in the early colonization stage of this species, which does not seem to be able to colonise waters deeper than 1 m in investigated sites, while it has been observed in significantly deeper waters in other European countries with a longer invasion history. Detecting the environmental parameters that most influence the growth of L. hexapetala becomes crucial both to identify the sites most at-risk of invasion in which to initiate timely monitoring actions for the species, and to be able to develop better management and control actions for this alien species in sites that have already been invaded.

Independent transitions to freshwater environments promote phenotypic divergence, not convergence, in stingrays.

Instances of convergent or parallel evolution provide a potent model system for exploring contingency and determinism in evolutionary biology. Likewise, the multiple, independent habitat transitions from saltwater to freshwater biomes offer opportunity for studying convergent evolution within and among different vertebrate lineages. For example, stingrays have invaded freshwater habitats multiple times across different continents, sometimes even several times within the same clade (e.g., Dasyatidae). We evaluated the frequency of saltwater-freshwater invasions in stingrays, compared ecological and phenotypic diversification among freshwater and saltwater lineages, and assessed the degree of convergence among freshwater species. Despite not being morphologically distinct from saltwater stingrays, freshwater stingrays do expand the margins of stingray morphological diversity. According to our data, trophic specialists occupied non-overlapping regions of morphospace, with piscivores and molluscivores being distinct from other diet guilds. Freshwater stingrays as a group did not strongly converge morphologically, neither did freshwater rays from different lineages which shared similar niches. These findings could be explained by there not being enough time for convergence to occur among more ancient and more recent freshwater lineages. Alternatively, the different ancestral bauplans of various freshwater ray lineages and weak selection on optimal phenotypes could promote contingency in the form of evolution along paths of least resistance.

A New Record of a Nonnative Bivalve Species in an Amazonian Environmental Protection Area: What Might Have Happened?

Corbicula fluminea is an Asian mussel that has aggressively invaded freshwater habitats worldwide, causing damage both economically and with the disappearance of native species. In Brazil, this species is present in almost all watersheds, including areas of Federal Conservation Units. In this context, the present work reported the first record of C. fluminea in the Carajás mosaic, Pará, Brazil, a Federal Conservation Area. Collections were made in September and February 2021 and May 2022. In total, 154 specimens of C. fluminea were collected. The size range of the specimens was 16 to 28 mm in shell length and 1.46 to 5.91 g in wet weight. This new record extends the distribution of this species in South America and in the tropical watersheds of Brazil and is the first record in federal protected areas, highlighting the need for large-scale knowledge of the environmental and economic changes that the species can cause.

Environment and Co-occurring Native Mussel Species, but Not Host Genetics, Impact the Microbiome of a Freshwater Invasive Species (Corbicula fluminea).

The Asian clam Corbicula fluminea (Family: Cyneridae) has aggressively invaded freshwater habitats worldwide, resulting in dramatic ecological changes and declines of native bivalves such as freshwater mussels (Family: Unionidae), one of the most imperiled faunal groups. Despite increases in our knowledge of invasive C. fluminea biology, little is known of how intrinsic and extrinsic factors, including co-occurring native species, influence its microbiome. We investigated the gut bacterial microbiome across genetically differentiated populations of C. fluminea in the Tennessee and Mobile River Basins in the Southeastern United States and compared them to those of six co-occurring species of native freshwater mussels. The gut microbiome of C. fluminea was diverse, differed with environmental conditions and varied spatially among rivers, but was unrelated to host genetic variation. Microbial source tracking suggested that the gut microbiome of C. fluminea may be influenced by the presence of co-occurring native mussels. Inferred functions from 16S rRNA gene data using PICRUST2 predicted a high prevalence and diversity of degradation functions in the C. fluminea microbiome, especially the degradation of carbohydrates and aromatic compounds. Such modularity and functional diversity of the microbiome of C. fluminea may be an asset, allowing to acclimate to an extensive range of nutritional sources in invaded habitats, which could play a vital role in its invasive success.

Simultaneous Expression of UV and Violet SWS1 Opsins Expands the Visual Palette in a Group of Freshwater Snakes.

Snakes are known to express a rod visual opsin and two cone opsins, only (SWS1, LWS), a reduced palette resulting from their supposedly fossorial origins. Dipsadid snakes in the genus Helicops are highly visual predators that successfully invaded freshwater habitats from ancestral terrestrial-only habitats. Here, we report the first case of multiple SWS1 visual pigments in a vertebrate, simultaneously expressed in different photoreceptors and conferring both UV and violet sensitivity to Helicops snakes. Molecular analysis and in vitro expression confirmed the presence of two functional SWS1 opsins, likely the result of recent gene duplication. Evolutionary analyses indicate that each sws1 variant has undergone different evolutionary paths with strong purifying selection acting on the UV-sensitive copy and dN/dS ∼1 on the violet-sensitive copy. Site-directed mutagenesis points to the functional role of a single amino acid substitution, Phe86Val, in the large spectral shift between UV and violet opsins. In addition, higher densities of photoreceptors and SWS1 cones in the ventral retina suggest improved acuity in the upper visual field possibly correlated with visually guided behaviors. The expanded visual opsin repertoire and specialized retinal architecture are likely to improve photon uptake in underwater and terrestrial environments, and provide the neural substrate for a gain in chromatic discrimination, potentially conferring unique color vision in the UV–violet range. Our findings highlight the innovative solutions undertaken by a highly specialized lineage to tackle the challenges imposed by the invasion of novel photic environments and the extraordinary diversity of evolutionary trajectories taken by visual opsin-based perception in vertebrates.

A Unique Expansion of the UV-Violet Visual Opsin Palette in a Group of Freshwater Snakes

Snakes are known to express up to three visual pigments, a rhodopsin and two cone opsins, SWS1 and LWS. Colubrid snakes in the genus Helicops have invaded freshwater habitats from an ancestral terrestrial-only condition. Here we report, for the first time in a tetrapod, the presence of two distinct SWS1 opsins simultaneously expressed in different photoreceptors, conferring both UV and violet sensitivity in a Helicops species. Molecular analysis and in vitro expression confirm the presence of two functional SWS1 copies, likely the result of a recent gene duplication. Evolutionary analyses indicate that each sws1 has undergone different evolutionary paths, with strong purifying selection acting on the UV-sensitive copy and neutrality on the violet-sensitive copy. Site-directed mutagenesis shows, for the first time in snakes, the functional role of a single amino acid substitution, Phe86Val, in the large spectral shift between UV and violet opsins. In addition, higher densities of photoreceptors and SWS cones in the ventral retina suggest improved acuity in the upper visual field possibly correlated with visually-guided behaviors. The expanded visual opsin repertoire and the specialized retinal architecture are likely to improve photon uptake in underwater and terrestrial environments, and provide the neural substrate for a gain in chromatic discrimination, potentially conferring unique color vision in the UV-violet range. Our findings highlight the innovative mechanisms undertaken by a highly specialized tetrapod lineage to tackle the challenges imposed by the invasion of novel photic environments and the extraordinary diversity of evolutionary trajectories taken by visual opsin- based perception in vertebrates.

Phenotypic divergence among threespine stickleback that differ in nuptial coloration.

By studying systems in their earliest stages of differentiation, we can learn about the evolutionary forces acting within and among populations and how those forces could contribute to reproductive isolation. Such an understanding would help us to better discern and predict how selection leads to the maintenance of multiple morphs within a species, rather than speciation. The postglacial adaptive radiation of the threespine stickleback (Gasterosteus aculeatus) is one of the best‐studied cases of evolutionary diversification and rapid, repeated speciation. Following deglaciation, marine stickleback have continually invaded freshwater habitats across the northern hemisphere and established resident populations that diverged innumerable times from their oceanic ancestors. Independent freshwater colonization events have yielded broadly parallel patterns of morphological differences in freshwater and marine stickleback. However, there is also much phenotypic diversity within and among freshwater populations. We studied a lesser‐known freshwater “species pair” found in southwest Washington, where male stickleback in numerous locations have lost the ancestral red sexual signal and instead develop black nuptial coloration. We measured phenotypic variation in a suite of traits across sites where red and black stickleback do not overlap in distribution and at one site where they historically co‐occurred. We found substantial phenotypic divergence between red and black morphs in noncolor traits including shape and lateral plating, and additionally find evidence that supports the hypothesis of sensory drive as the mechanism responsible for the evolutionary switch in color from red to black. A newly described third “mixed” morph in Connor Creek, Washington, differs in head shape and size from the red and black morphs, and we suggest that their characteristics are most consistent with hybridization between anadromous and freshwater stickleback. These results lay the foundation for future investigation of the underlying genetic basis of this phenotypic divergence as well as the evolutionary processes that may drive, maintain, or limit divergence among morphs.

Selection, Linkage, and Population Structure Interact To Shape Genetic Variation Among Threespine Stickleback Genomes.

The outcome of selection on genetic variation depends on the geographic organization of individuals and populations as well as the organization of loci within the genome. Spatially variable selection between marine and freshwater habitats has had a significant and heterogeneous impact on patterns of genetic variation across the genome of threespine stickleback fish. When marine stickleback invade freshwater habitats, more than a quarter of the genome can respond to divergent selection, even in as little as 50years. This process largely uses standing genetic variation that can be found ubiquitously at low frequency in marine populations, can be millions of years old, and is likely maintained by significant bidirectional gene flow. Here, we combine population genomic data of marine and freshwater stickleback from Cook Inlet, Alaska, with genetic maps of stickleback fish derived from those same populations to examine how linkage to loci under selection affects genetic variation across the stickleback genome. Divergent selection has had opposing effects on linked genetic variation on chromosomes from marine and freshwater stickleback populations: near loci under selection, marine chromosomes are depauperate of variation, while these same regions among freshwater genomes are the most genetically diverse. Forward genetic simulations recapitulate this pattern when different selective environments also differ in population structure. Lastly, dense genetic maps demonstrate that the interaction between selection and population structure may impact large stretches of the stickleback genome. These findings advance our understanding of how the structuring of populations across geography influences the outcomes of selection, and how the recombination landscape broadens the genomic reach of selection.

Evolutionary structure and timing of major habitat shifts in Crocodylomorpha

Extant crocodylomorphs are semiaquatic ambush predators largely restricted to freshwater or estuarine environments, but the group is ancestrally terrestrial and inhabited a variety of ecosystems in the past. Despite its rich ecological history, little effort has focused on elucidating the historical pattern of ecological transitions in the group. Traditional views suggested a single shift from terrestrial to aquatic in the Early Jurassic. However, new fossil discoveries and phylogenetic analyses tend to imply a multiple-shift model. Here we estimate ancestral habitats across a comprehensive phylogeny and show at least three independent shifts from terrestrial to aquatic and numerous other habitat transitions. Neosuchians first invade freshwater habitats in the Jurassic, with up to four subsequent shifts into the marine realm. Thalattosuchians first appear in marine habitats in the Early Jurassic. Freshwater semiaquatic mahajangasuchids are derived from otherwise terrestrial notosuchians. Within nearly all marine groups, some species return to freshwater environments. Only twice have crocodylomorphs reverted from aquatic to terrestrial habitats, both within the crown group. All living non-alligatorid crocodylians have a keratinised tongue with salt-excreting glands, but the lack of osteological correlates for these adaptations complicates pinpointing their evolutionary origin or loss. Based on the pattern of transitions to the marine realm, our analysis suggests at least four independent origins of saltwater tolerance in Crocodylomorpha.

The Legs Have It: In Situ Expression of Ion Transporters V-Type H(+)-ATPase and Na(+)/K(+)-ATPase in the Osmoregulatory Leg Organs of the Invading Copepod Eurytemora affinis.

The copepod Eurytemora affinis has an unusually broad salinity range, as some populations have recently invaded freshwater habitats independently from their ancestral saline habitats. Prior studies have shown evolutionary shifts in ion transporter activity during freshwater invasions and localization of ion transporters in newly discovered "Crusalis organs" in the swimming legs. The goals of this study were to localize and quantify expression of ion transport enzymes V-type H(+)-ATPase (VHA) and Na(+)/K(+)-ATPase (NKA) in the swimming legs of E. affinis and determine the degree of involvement of each leg in ionic regulation. We confirmed the presence of two distinct types of ionocytes in the Crusalis organs. Both cell types expressed VHA and NKA, and in the freshwater population the location of VHA and NKA in ionocytes was, respectively, apical and basal. Quantification of in situ expression of NKA and VHA established the predominance of swimming leg pairs 3 and 4 in ion transport in both saline and freshwater populations. Increases in VHA expression in swimming legs 3 and 4 of the freshwater population (in fresh water) relative to the saline population (at 15 PSU) arose from an increase in the abundance of VHA per cell rather than an increase in the number of ionocytes. This result suggests a simple mechanism for increasing ion uptake in fresh water. In contrast, the decline in NKA expression in the freshwater population arose from a decrease in ionocyte area in legs 4, likely resulting from decreases in number or size of ionocytes containing NKA. Such results provide insights into mechanisms of ionic regulation for this species, with added insights into evolutionary mechanisms underlying physiological adaptation during habitat invasions.

Evolutionary mechanisms of habitat invasions, using the copepod Eurytemora affinis as a model system.

The study of the copepod Eurytemora affinis has provided unprecedented insights into mechanisms of invasive success. In this invited review, I summarize a subset of work from my laboratory to highlight key insights gained from studying E.affinis as a model system. Invasive species with brackish origins are overrepresented in freshwater habitats. The copepod E.affinis is an example of such a brackish invader, and has invaded freshwater habitats multiple times independently in recent years. These invasions were accompanied by the evolution of physiological tolerance and plasticity, increased body fluid regulation, and evolutionary shifts in ion transporter (V-type H(+) ATPase, Na(+), K(+)-ATPase) activity and expression. These evolutionary changes occurred in parallel across independent invasions in nature and in laboratory selection experiments. Selection appears to act on standing genetic variation during invasions, and maintenance of this variation is likely facilitated through 'beneficial reversal of dominance' in salinity tolerance across habitats. Expression of critical ion transporters is localized in newly discovered Crusalis leg organs. Increased freshwater tolerance is accompanied by costs to development time and greater requirements for food. High-food concentration increases low-salinity tolerance, allowing saline populations to invade freshwater habitats. Mechanisms observed here likely have relevance for other taxa undergoing fundamental niche expansions.

Freshwater habitat use by a moray eel species, Gymnothorax polyuranodon, in Fiji shown by otolith microchemistry

Freshwater eels of the Anguillidae are diadromous because they migrate between ocean and freshwater environments, but other anguilliform fishes are generally considered to be strictly marine species. A few marine eels of the Muraenidae and Ophichthidae have occasionally been found in freshwater or estuaries, indicating that anguillids are not the only anguilliform eels that can use freshwater in some parts of the world. The moray eel Gymnothorax polyuranodon is one species that is known to be present in freshwater in the Indo-Pacific, but its life history is unknown. One way to evaluate what types of habitats are used by fishes is to determine the ratio of strontium (Sr) to calcium (Ca) in their otoliths, because this can show if they have used freshwater or saltwater environments. To evaluate the patterns of freshwater use by this unusual species of marine eel, the otolith Sr/Ca ratios of four G. polyuranodon (275–344 mm) caught in a freshwater stream of Fiji were analyzed. The consistently low Sr/Ca values (0–4) indicated upstream movement after settlement and freshwater or estuarine residence of all four individuals. These eels did not appear to have entered freshwater just for a short time period, which is consistent with other reports that this species is present in estuarine and freshwater habitats. This suggests that G. polyuranodon may be a catadromous species of marine eel. The similarities and differences between the life histories of anguillid eels and the few marine eels that have evolved the ability to invade freshwater habitats is discussed in relation to the evolutionary origin of diadromy in anguilliform fishes that originated in the marine environment.

Without Gills: Localization of Osmoregulatory Function in the CopepodEurytemora affinis

The Pancrustacea, which include crustaceans and hexapods, have successfully colonized marine, freshwater, and terrestrial habitats. While members of the class Malacostraca (e.g., crabs, shrimp) often display immense osmoregulatory capacities, more basally branching crustaceans (e.g., copepods, branchiopods) tend to possess less-specialized osmoregulatory structures that have been poorly characterized. Remarkably, some of these more basal taxa have also colonized diverse habitats. For instance, the copepod Eurytemora affinis has recently invaded freshwater habitats multiple times independently but lack obvious osmoregulatory structures. To explore localization of ion exchange, we performed silver staining, immunohistochemical staining, and transmission electron microscopy. Our results revealed localization of ion transport within the maxillary glands and on four pairs of swimming legs. Silver staining revealed ion exchange at the maxillary pores and on the endopods and exopods of swimming legs P1 through P4. Immunohistochemical assays localized ion transport enzymes V-type H(+)-ATPase and Na(+)/K(+)-ATPase in the maxillary glands and swimming legs as well. Finally, transmission electron microscopy identified specialized ionocytes within these anatomical regions. These investigations uncovered novel osmoregulatory structures at the swimming legs, which we designate the "Crusalis organs." Our findings identified specific tissues specialized for ion transport, potentially enabling this small crustacean to rapidly transition into freshwater habitats.

The population structure and recent colonization history of Oregon threespine stickleback determined using restriction-site associated DNA-sequencing.

Understanding how genetic variation is partitioned across genomes within and among populations is a fundamental problem in ecological and evolutionary genetics. To address this problem, we studied the threespine stickleback fish, which has repeatedly undergone parallel phenotypic and genetic differentiation when oceanic fish have invaded freshwater habitats. While significant evolutionary genetic research has been performed using stickleback from geographic regions that have been deglaciated in the last 20 000 years, less research has focused on freshwater populations that predate the last glacial maximum. We performed restriction-site associated DNA-sequencing (RAD-seq) based population genomic analyses on stickleback from across Oregon, which was not glaciated during the last maximum. We sampled stickleback from coastal, Willamette Basin and central Oregon sites, analysed their genetic diversity using RAD-seq, performed structure analyses, reconstructed their phylogeographic history and tested the hypothesis of recent stickleback introduction into central Oregon, where incidence of this species was only recently documented. Our results showed a clear phylogeographic break between coastal and inland populations, with oceanic populations exhibiting the lowest levels of divergence from one another. Willamette Basin and central Oregon populations formed a clade of closely related populations, a finding consistent with a recent introduction of stickleback into central Oregon. Finally, genome-wide analysis of genetic diversity (π) and correlations of alleles within individuals in subpopulations (FIS) supported a role for introgressive hybridization in coastal populations and a recent expansion in central Oregon. Our results exhibit the power of next-generation sequencing genomic approaches such as RAD-seq to identify both historical population structure and recent colonization history.

Regulation of Muscle Hydration Upon Hypo‐ or Hyper‐Osmotic Shocks: Differences Related to Invasion of the Freshwater Habitat by Decapod Crustaceans

Decapod crustaceans have independently invaded freshwater habitats from the sea/estuaries. Tissue hydration mechanisms are necessary for the initial stages of habitat transitions but can be expected to diminish, as the capacity for extracellular homeostasis increases in hololimnetic species. Six decapod species have been compared concerning the maintenance of muscle hydration in vitro: Hepatus pudibundus (marine); Palaemon pandaliformis (estuarine resident), Macrobrachium acanthurus (freshwater diadromous), and the three hololimnetic Macrobrachium potiuna, Dilocarcinus pagei, and Aegla parana. The effects of inhibitors of potassium channels (barium chloride) and NKCC (furosemide) were evaluated under isosmotic, and respectively hypo- (50% below iso) or hyper- (50% above iso) conditions. There was high muscle hydration control in H. pudibundus with a possible role of NKCC in isosmotic conditions. Shrimps consistently showed small deviations in muscle hydration under anisosmotic conditions; P. pandaliformis has shown evidence of the presence of NKCC; M. potiuna was the species less affected by both inhibitors, under iso- or anisosmotic conditions. In the two hololimnetic crab species, both independent long-time inhabitants of freshwater, while the capacity to deal with hyper-osmotic shock is decreased, the capacity to deal with hyposmotic shock is retained, possibly because of hemolymph dilution during molting in fresh water. D. pagei apparently depends on potassium channels for volume recovery after swelling, whereas A. parana shows some dependence on NKCC to minimize volume loss in hyper-osmotic conditions. Although no molecular screening techniques have been tried here, data point to distinct cell/tissue transport mechanisms acting upon hydration/volume challenges in decapods of different habitats and lineages.

Feasting in fresh water: impacts of food concentration on freshwater tolerance and the evolution of food × salinity response during the expansion from saline into fresh water habitats

Saline to freshwater invasions have become increasingly common in recent years. A key hypothesis is that rates of freshwater invasions have been amplified in recent years by increased food concentration, yet this hypothesis has remained unexplored. We examined whether elevated food concentration could enhance freshwater tolerance, and whether this effect evolves following saline to freshwater invasions. We examined physiological response to salinity and food concentration in a 2 × 2 factorial design, using ancestral brackish and freshwater invading populations of the copepod Eurytemora affinis. We found that high food concentration significantly increases low-salinity tolerance. This effect was reduced in the freshwater population, indicating evolution following the freshwater invasion. Thus, ample food could enable freshwater invasions, allowing subsequent evolution of low-salinity tolerance even under food-poor conditions. We also compared effects of food concentration on freshwater survival between two brackish populations from the native range. Impacts of food concentration on freshwater survival differed between the brackish populations, suggesting variation in functional properties affecting their propensity to invade freshwater habitats. The key implication is that high food concentration could profoundly extend range expansions of brackishwater species into freshwater habitats, potentially allowing for condition-specific competition between saline invaders and resident freshwater species.

Comparison of salinity tolerance and osmoregulation in two closely related species of blennies from different habitats

This study compares osmoregulatory capabilities of two closely related blennies inhabiting different habitats: Salaria pavo which inhabits marine rocky coasts in the Mediterranean and the eastern coasts of the Atlantic Ocean, and Salaria fluviatilis which inhabits freshwater habitats around the Mediterranean, both in rivers connected to the sea and in lakes that for a long time have been separated from the sea. Fishes for this study were collected in Lake Kinneret (Sea of Galilee) (S. fluviatilis) and Habonim beach, Israel, East Mediterranean coast (S. pavo). Both species tolerated salinities of 0, 40 and 100% seawater (0, 14.4 and 36 ppt sea salt respectively). Acclimation for a period of three months to freshwater (S. pavo) and seawater (S. fluviatilis) did not affect survival rates. After acclimation, total body water content of S. fluviatilis remained unaffected by experimental salinities but increased in S. pavo as salinity decreased. The hematocrit of both species increased as salinity increased. Plasma osmolality and Na+ concentrations were unaffected by experimental acclimation salinities in S. fluviatilis, but significantly decreased in S. pavo as salinity decreased. The results indicate that S. fluviatilis is able to osmoregulate both in fresh and seawater. S. pavo, living in seawater, while able to tolerate freshwater environment, fails to maintain constant extracellular fluid concentration. It is suggested that S. pavo, or its marine ancestor, has euryhaline capabilities that enabled it to invade freshwater habitats. S. fluviatilis, as the evolutionary result of this invasion, is thus better adapted to freshwater environment, but did not lose its ability to live in seawater.

Observations on the morphology and behaviour ofChilina fluctuosaGray (Chilinidae), with a discussion on the early evolution of pulmonate gastropods

An account is provided of (i) the general morphology, (ii) the vascular, nervous and feeding systems, and (iii) aspects of the behaviour of a representative species of the pulmonate genusChilina. Examination of this genus has aided in understanding the pattern of early evolution of freshwater pulmonates from their marine prosobranch ancestors and of their divergence from opisthobranchs. The gross morphology ofChilinais adapted to ploughing through the surface layers of soft substrata. AlthoughC. fluctuosawas found on rocks, it was nevertheless capable of burrowing into sand, using a stereotyped digging cycle. Streamlining to facilitate burrowing was achieved by nuchal shortening and by a ‘detorsion’ of the mantle complex as in opisthobranchs. The nuchal shortening incurred loss of a major vascular pathway which, in monotocardian prosobranchs, drains the head-foot. ‘Detorsion’, however, provided a substitute which incorporates the anterior roof of the mantle cavity where, inChilina, the precursor of the rich pulmonary plexus characteristic of later pulmonates may be discerned. The suprapallalial vascular system is described in detail. In contrast, it is apparent that increased reliance on burrowing in opisthobranchs brought about loss of the anterior region of the mantle cavity, and thus any potential for development of a plexus in that region. Enclosure of the mantle cavity, which clearly pre-adapted the mantle complex to function also as a ‘lung’, was necessary to reduce clogging of the mantle cavity by particulate material. Only water is held in the cavity ofChilina flutuosa, whose habitat is rivers, but both air and water may be held in the mantle complex of other species found in Chile. The nervous system is extremely similar to that of the primitive opisthrobranchActeon, but the posterior section of the visceral loop is shorter owing to greater nuchal reduction. The loop is uncrossed, though the supraoesophageal ganglion lies only a little right of the suboesophageal ganglion. The musculature of the buccal mass is extremely similar to that ofLymnaeaandPlanorbarius, and is designed for moving and tensing a broad radular ribbon. It is argued that the buccal morphology characteristic of these snails evolved to manipulate quantities of particulate material during the early infaunal phase, and only later was used to great effect in both microphagous and macrophagous feeding. Collectively, the observations made onC. flluctuosa(and also on estuarine species found in Chile) support the contention that the Basommatophora invaded freshwater habitats via estuarine niches. Continuity during this progression was apparently provided by soft substrata, and probably by diatoms as a food source. Previous arguments purporting a terrestrial or semi-terrestrial origin for the limnic basommatophorans are refuted.